Urokinase-type plasminogen activator transgenic mouse

ABSTRACT

The present invention provides a mouse with liver damage, having a high degree of damage against the mouse&#39;s original hepatocytes while having a uPA gene in a heterozygous form, and a method for efficiently preparing the mouse. Specifically, the method for preparing a mouse with liver damage having the uPA gene in a heterozygous form comprises the following steps of: 
     (i) transforming mouse ES cells with a DNA fragment containing a liver-specific promoter/enhancer and cDNA that encodes a urokinase-type plasminogen activator operably linked under the control thereof; 
     (ii) injecting the transformed mouse ES cells obtained in step (i) into a host embryo; 
     (iii) transplanting the host embryo obtained in step (ii) via the injection of the ES cells into the uterus of a surrogate mother mouse, so as to obtain a chimeric mouse; and 
     (iv) crossing the chimeric mice obtained in step (iii), so as to obtain a transgenic mouse in which the DNA fragment is introduced in a heterozygous form.

TECHNICAL FIELD

The present invention relates to a mouse with liver damage, which isprepared by introducing a DNA fragment that contains a liver-specificpromoter/enhancer and cDNA encoding an urokinase-type plasminogenactivator operably linked under the control thereof, into ES cells andthen using the ES cells, wherein the DNA fragment is introduced in aheterozygous form.

BACKGROUND ART

Experimentation using human cells is generally desired to study humandiseases. In particular, studies of diseases, in which manydrug-metabolizing enzymes confirmed to have species specificity,viruses, the hosts of which are limited to humans, and the like areinvolved, require to use human cells, and particularly humanhepatocytes. However, the supply of human hepatocytes is limited and invitro proliferation of human hepatocytes while keeping theirdifferentiation status is very difficult. The use of in vivo environmentis relatively efficient for the proliferation of human hepatocytes.Specifically, a gene accelerating the death of mouse hepatocytes isintroduced into mice that have been produced from immunodeficient miceas the genetic background to produce transgenic mice, human hepatocytesare transplanted into the transgenic mice, and then human hepatocytesare proliferated. In this manner, the replacement of most mousehepatocytes by human hepatocytes has been attempted.

Liver disease caused by the infection of human liver with viruses is adisease difficult to treat in recent years in medical practice. Animalspecies susceptible to these viruses that infect human hepatocytes arelimited to humans and chimpanzees. Tests using human hepatocytes arerequired to develop remedies against these viral infections. Also,hepatocytes play important roles in drug metabolism. Elucidation of themetabolic pathways of individual drugs in humans is considered to leadto the development of new pharmaceutical products. However, speciesspecificity is present in many drug-metabolizing enzymes, and thuselucidation of the drug metabolic pathways in humans requires to conducttests using human hepatocytes.

Regarding Hepatitis C virus (HCV), about 1500,000 carriers of HepatitisC virus (HCV carriers), and about 400,000 to 500,000 patients other thanthese carriers are estimated to be treated in Japan. The number ofchronic hepatitis C patients receiving interferon administration is saidto be annually 30,000 to 40,000. In these days, new antiviral agentstargeting various sites of viral genome are under development. However,the advancement thereof is significantly inhibited because of the lackof reliable HCV animal models with high reproducibility. This can besaid for not only HCV, but also other types of viral hepatitis such ashepatitis type B virus (HBV). Hosts for these viruses are only humansand chimpanzees. Therefore, development of small model animals producedby replacing human hepatocytes by a host's hepatocytes is desired forlarge-scale development and study of antiviral agents using animals.

Fatty liver is developed due to the accumulation of neutral fat in theliver. In recent years, the incidence of non-alcoholic steatohepatitis(NASH) that is hepatitis resulting from the accumulation of fat in theliver is increasing. This disease may proceed to diseases with poorprognosis such as chronic hepatitis, hepatic cirrhosis, andhepatocellular carcinoma. Meanwhile, the absence of effective remediesagainst such liver diseases has been suggested (Non-patent Literature1). The development of such remedies also requires the presence ofoptimum animal models.

If the use of model animals having human hepatocytes as a result ofreplacement becomes possible for the study of the above diseases, thiswill contribute to many studies for drug development. However, thepreparation of the model animals requires efficient proliferation ofhuman hepatocytes after transplantation thereof into host animals andsuccessful replacement thereof by the host's hepatocytes.

Several examples of transplantation of human hepatocytes into transgenicmice have been reported, wherein human hepatocytes are transplanted intothe transgenic mice in which an urokinase-type plasminogen activator(hereinafter, referred to as “uPA”) gene is expressedliver-specifically, so as to damage mouse hepatocytes. uPA transgenicmice prepared using the genomic sequence of uPA (Non-patent Literature2) and uPA transgenic mice prepared using the cDNA of uPA (Non-patentLiterature 3) have been reported. All of these uPA transgenic mice arerequired to have the uPA gene in a homozygous form, since theengraftment of transplanted human hepatocytes is difficult when the micehave the uPA gene in a heterozygous form. However, the preparation oftransgenic mice having the uPA gene in a homozygous form requires atleast two generations and at least 6 months. Moreover, homozygous miceare obtained in a proportion of about only 25% with respect to the totalnumber of the thus obtained mice. It has been difficult to preparetransgenic mice having a large quantity of the uPA gene in a homozygousform within a short period. It has also been difficult to prepare across-bred line with another transgenic mouse due to a similar reason.Moreover, in transgenic mice produced using a conventional uPA genomicsequence, the recombination of the uPA gene introduced into the livertakes place over time, and the loss of the uPA gene is observed. Sincemouse cells lacking the uPA gene regenerate hepatocytes again, it hasbeen difficult for human hepatocytes to engraft after transplantationthereof into heterozygous mice. Furthermore, in homozygous mice, mousehepatocytes are regenerated due to the loss of the uPA gene, and thus agradual decrease in human hepatocytes that have engrafted is frequentlyobserved among mice. Hence, uPA transgenic mice that can be producedefficiently in large quantity and enables easy preparation of across-bred line with another transgenic mouse have been desperatelydesired in the art.

PRIOR ART LITERATURE Non-Patent Literature

-   Non-patent Literature 1 N Engl 7 Med. 346:1221-31 (2002)-   Non-patent Literature 2 Cell 66: 245-256 (1991)-   Non-patent Literature 3 BBRC 377: 248-252 (2008)

SUMMARY OF THE INVENTION

The present invention provides mice with liver damage, having a highdegree of damage to the original mouse hepatocytes while having the uPAgene in a heterozygous form, and a method for efficiently preparing themice.

As a result of intensive studies to achieve the above object, thepresent inventors have discovered that transgenic mice having a highdegree of damage to the original mouse hepatocytes while having the uPAgene in a heterozygous form can be efficiently prepared by introducing aDNA fragment that contains a liver-specific promoter/enhancer and cDNAencoding uPA operably linked under the control thereof, into mouse EScells and then using the ES cells. The present inventors have alsodiscovered that no or almost no loss of the introduced uPA gene takesplace over time in the transgenic mice.

The present inventors have further discovered that human hepatocytestransplanted into immunodeficient mice with liver damage can engraft,which are prepared using the above transgenic mice.

The present invention is based on these findings. Specifically, thepresent invention encompasses the following [1] to [14].

[1] A method for preparing a mouse with liver damage, which has an uPAgene in a heterozygous form, comprising the following steps of:

(i) transforming mouse ES cells with a DNA fragment containing aliver-specific promoter/enhancer and cDNA that encodes a urokinase-typeplasminogen activator operably linked under the control thereof;

(ii) injecting the transformed mouse ES cells obtained in step (i) intoa host embryo;

(iii) transplanting the host embryo obtained in step (ii) via theinjection of the ES cells into the uterus of a surrogate mother mouse,so as to obtain a chimeric mouse; and

(iv) crossing the chimeric mice obtained in step (iii), so as to obtaina transgenic mouse in which the DNA fragment is introduced in aheterozygous form.

[2] The method of [1], further comprising step (v) of obtaining atransgenic mouse in which the serum ALT level of the 2- to 3-week-oldtransgenic mouse is 30 (Karmen unit) or more.

[3] The method of [1] or [2], wherein the liver-specific promoter is analbumin promoter.

[4] A mouse with liver damage prepared by the method of [1] to [3] and aportion thereof.

[5] An immunodeficient mouse with liver damage, which is obtained bycrossing the mouse with liver damage of [4] with a SCID mouse.

[6] A method for preparing a chimeric mouse characterized by having achimeric liver containing human hepatocytes, comprising transplantinghuman hepatocytes into the immunodeficient mouse with liver damage of[5].

[7] A chimeric mouse prepared by the method of [6], which has a chimericliver containing human hepatocytes.

[8] A chimeric mouse, which is immunodeficient, has a DNA fragmentcontaining a liver-specific promoter/enhancer and cDNA that encodes aurokinase-type plasminogen activator operably linked under the controlthereof, in a heterozygous form, and has a chimeric liver containinghuman hepatocytes.[9] The chimeric mouse of [7] or [8], wherein human hepatocytes accountfor at least 10% of all hepatocytes in the chimeric liver.[10] The chimeric mouse of [7] or [8], wherein the human hepatocytesretain their functions and properties for at least 2 weeks in thechimeric liver.[11] A method for screening for a substance that affects human liverfunctions, comprising the following steps (a) to (c) of:(a) administering a test substance to the chimeric mouse of any one of[7] to [10];(b) measuring one or more values selected from the group consisting ofthe human albumin concentration, the body weight curve, theliver-weight-to-body-weight ratio, the total albumin level, the totalprotein level, the ALT level, the AST level, and the total bilirubinlevel in the chimeric mouse to which the test substance is administeredin (a); and(c) selecting a test substance that causes an increase or a decrease inany one or more of the human albumin concentration, the body weightcurve, the liver-weight-to-body-weight ratio, the total albumin level,the total protein level, the ALT level, the AST level, and the totalbilirubin level measured in (b), compared with the human albuminconcentration, the body weight curve, the liver-weight-to-body-weightratio, the total albumin level, the total protein level, the ALT level,the AST level, and the total bilirubin level of the chimeric mouse towhich no test substance is administered.[12] A method for evaluating the toxicity of a test substance againsthuman hepatocytes, comprising the following steps (a) to (c) of:(a) administering a test substance to the chimeric mouse of any one of[7] to [10];(b) measuring one or more values selected from the group consisting ofthe human albumin concentration, the body weight curve, theliver-weight-to-body-weight ratio, the total albumin level, the totalprotein level, the ALT level, the AST level, and the total bilirubinlevel in the chimeric mouse to which the test substance is administeredin (a); and(c) evaluating the effect of the test substance on human hepatocytesusing, as an indicator, an increase or a decrease in any one or more ofthe human albumin concentration, the body weight curve, theliver-weight-to-body-weight ratio, the total albumin level, the totalprotein level, the ALT level, the AST level, and the total bilirubinlevel measured in (b), compared with the human albumin concentration,the body weight curve, the liver-weight-to-body-weight ratio, the totalalbumin level, the total protein level, the ALT level, the AST level,and the total bilirubin level of the chimeric mouse to which no testsubstance is administered.[13] A method for screening for a substance effective for treatment ofviral hepatitis, comprising the following steps (a) to (d) of:(a) inoculating a hepatitis virus into the chimeric mouse of any one of[7] to [10];(b) administering a test substance to the chimeric mouse inoculated withthe hepatitis virus in (a);(c) measuring one or more values selected from the group consisting ofthe human albumin concentration, the body weight curve, theliver-weight-to-body-weight ratio, the total albumin level, the totalprotein level, the ALT level, the AST level, the total bilirubin level,the viral load, and the amount of a virus-derived protein of thechimeric mouse to which the test substance is administered in (b); and(d) selecting a test substance causing a change in any one or more ofthe human albumin concentration, the body weight curve, theliver-weight-to-body-weight ratio, the total albumin level, the totalprotein level, the ALT level, the AST level, the total bilirubin level,the viral load, and the amount of a virus-derived protein measured in(c), compared with the human albumin concentration, the body weightcurve, the liver-weight-to-body-weight ratio, the total albumin level,the total protein level, the ALT level, the AST level, the totalbilirubin level, the viral load, and the amount of a virus-derivedprotein in the chimeric mouse to which no test substance isadministered.[14] The method of [13], wherein the hepatitis virus is hepatitis type Avirus, hepatitis type B virus, hepatitis type C virus, hepatitis type Dvirus, or hepatitis type E virus.

This description includes all or part of the contents as disclosed inthe description and/or drawings of Japanese Patent Application No.2012-102814, from which the present application claims the priority.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one color drawing.Copies of this patent or patent application publication with colordrawing will be provided by the USPTO upon request and payment of thenecessary fee.

FIG. 1 is a schematic view showing an uPA gene insertion vector forfertilized eggs, “mAlb uPAInt2”. SV40 pA: SV40 polyA signal; mAlbPro/En:mouse albumin enhancer/promoter; uPA cDNA: the ORF portion of mouse uPA;exon-intron-exon: the 2^(nd) exon, intron, and the 3^(rd) exon of rabbitβ globin; polyA+About 50 bp: polyA signal in the 3^(rd) exon of rabbit βglobin.

FIG. 2 is a schematic view showing an uPA gene insertion vector for EScells, “mAlb uPAInt2ES”. SV40 pA: SV40 polyA signal; mAlbPro/En: mousealbumin enhancer/promoter; uPA cDNA: the ORF portion of mouse uPA;exon-intron-exon: the 2^(nd) exon, intron, and the 3^(rd) exon of rabbitβ-globin; polyA+About 50 bp: polyA signal in the 3^(rd) exon of rabbitβ-globin.

FIG. 3 shows the results of measuring, the ALT levels and so on in uPAtransgenic mice prepared via ES cells.

FIG. 4 shows the results of measuring human albumin concentrations inmouse blood (top) and body weights (bottom) of #1C2 mice (up to 14 weeksold) after transplantation of human hepatocytes into the mice. The solidlines denote homozygous mice and dotted lines denote heterozygous mice.

FIG. 5 shows the results of measuring human albumin concentrations inmouse blood (top) and body weights (bottom) of #2C7 mice (up to 14 weeksold) after transplantation of human hepatocytes into the mice. The solidlines denote homozygous mice and dotted lines denote heterozygous mice.

FIG. 6 shows the immunostaining images of chimeric mouse liver sectionsprepared using #1C2 homozygous, #1C2 heterozygous, and #2C7 homozygousmice immunostained with a human cytokeratin 8/18 antibody.

FIG. 7 shows the results of measuring the replacement rates in thelivers of chimeric mice prepared using 14-week-old (top) and 30-week-old(bottom) #1C2 homozygous, #1C2 heterozygous, and 2C7 homozygous mice andhuman albumin concentrations in the mouse blood.

FIG. 8-1 shows human albumin concentrations in mouse blood (left) beforeHCV inoculation and each viral copy number (right) in mouse serum afterinoculation of chimeric mice prepared using #1C2 homozygous and #1C2heterozygous mice. Solid lines denote homozygous mice and dotted linesdenote heterozygous mice.

FIG. 8-2 shows human albumin concentrations in mouse blood (left) beforeHBV inoculation and each viral copy numbers (right) in mouse serum afterinoculation of chimeric mice prepared using #1C2 homozygous and #1C2heterozygous mice. Solid lines denote homozygous mice and dotted linesdenote heterozygous mice.

FIG. 9-1 shows human albumin concentrations in mouse blood (left) beforeHCV inoculation and HCV copy numbers (right) in mouse serum afterinoculation of chimeric mice prepared using #2C7 homozygous mice.

FIG. 9-2 shows human albumin concentrations in mouse blood (left) beforeHBV inoculation and HBV copy numbers (right) in mouse serum afterinoculation of chimeric mice prepared using #2C7 homozygous mice.

FIG. 10 shows the results of measuring human albumin concentrations inmouse blood (top) and body weights (bottom) of #1C2 mice (up to 30 weeksold) after transplantation of human hepatocytes into the mice. Solidlines denote homozygous mice and dotted lines denote heterozygous mice.

FIG. 11 shows the results of measuring human albumin concentrations inmouse blood (top) and body weights (bottom) of #2C7 mice (up to 30 weeksold) after transplantation of human hepatocytes into the mice. Solidlines denote homozygous mice and dotted lines denote heterozygous mice.

MODES FOR CARRYING OUT THE INVENTION

The present invention will be described below in detail.

The mouse with liver damage of the present invention has a DNA fragmentcontaining a liver-specific promoter/enhancer and cDNA that encodes aurokinase-type plasminogen activator operably linked under the controlthereof, in a heterozygous form, whereby uPA is expressedliver-specifically, and at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95% or more of original mouse liver cells (particularly,hepatocytes) are damaged, the proliferation thereof is suppressed,and/or the cells are necrotized.

The mouse with liver damage of the present invention has a high degreeof damage against original mouse hepatocytes while having the uPA genein a heterozygous form, and thus are not required to have the uPA genein a homozygous form unlike conventionally known uPA transgenic mice.

The mouse with liver damage of the present invention can be prepared onthe basis of a conventionally known method for preparing transgenicanimals (Proc. Natl. Acad. Sci. U.S.A. 77: 7380-7384 (1980)) byintroducing a DNA fragment containing a liver-specific promoter/enhancerand cDNA that encodes uPA operably linked under the control thereof intomouse ES cells and then using the thus obtained ES cells.

The term “promoter/enhancer” refers to DNA having a sequence capable ofproviding the functions of both the promoter and the enhancer.

Examples of a “liver-specific promoter” include, but are notparticularly limited to, as long as it can induce the expression of agene ligated to the 3′ side in a liver-specific manner, an albuminpromoter, an α-fetoprotein promoter, an α₁-anti-trypsin promoter, atransferrin transthyretin promoter, a serum amyloid A promoter, atransthyretin promoter, and a hepatocyte nuclear factor 6 (HNF-6)promoter. A preferable example thereof is an albumin promoter.

The “liver-specific promoter/enhancer” may be any one of an endogenouspromoter/enhancer, an exogenous promoter/enhancer, a promoter/enhancerof the same species, a promoter/enhancer of a different species, anartificial promoter/enhancer, as long as it enables the expression of atarget gene liver-specifically. Preferably, a mouse-derivedpromoter/enhancer is used. A mouse-derived liver-specificpromoter/enhancer is known in the art. For example, an albuminpromoter/enhancer can be used. A mouse-derived albumin promoter/enhanceris known (Herbst R S et al, Proc Natl Acad Sci U.S.A. 1989 March; 86(5): 1553-7; Heckel J L et al., Cell 1990 August 10; 62(3): 447-56), andcan be obtained by performing PCR using primers specific to the albuminpromoter/enhancer and a mouse genomic library as a template.

uPA-encoding cDNA may be any one of endogenous cDNA, exogenous cDNA,cDNA of the same species, and cDNA of a different species. Preferably,mouse-derived cDNA is used. The uPA-encoding cDNA can be obtained by ageneral technique known by persons skilled in the art, specifically byperforming reverse transcription PCR using RNA extracted from the liveras a template and primers specific to an uPA-encoding gene. TheuPA-encoding gene was registered under Accession No. NM008873 in theabove published database. In the present invention, the gene informationcan be used (in this Description, the uPA-encoding gene is representedby SEQ ID NO: 11). In addition, in the Description, the term “uPA gene”described in the present invention refers to uPA-encoding cDNA. Theseterms can be used interchangeably.

The term “a liver-specific promoter/enhancer and cDNA that encodes uPAoperably linked under the control thereof” means that uPA-encoding cDNAis arranged so that uPA is expressed under the control of theliver-specific promoter/enhancer.

The DNA fragment containing a liver-specific promoter/enhancer and cDNAthat encodes uPA operably linked under the control thereof is introducedinto ES cells (embryonic stem cells).

The DNA fragment can be introduced into ES cells by a calcium phosphatemethod, an electrical pulse method, a lipofection method, an aggregationmethod, a microinjection method, a particle gun method, a DEAE-dextranmethod, or the like (examples thereof are not limited thereto).

ES cells prepared by introducing the DNA fragment can be cultured exvivo, so that cells into which the DNA fragment has been introducedsuccessfully and/or cells in which the introduced DNA fragment has notbeen lost can be screened for. Next, the thus obtained ES cells areinjected into a host embryo, and preferably a mouse blastocyst, theresultant is transplanted into the uterine horn of a surrogate mothermouse for generation, and thus transgenic mice (chimeric mice) are born.As a surrogate mother mouse, in general, a female pseudopregnant mouseproduced by crossing with a male mouse subjected to vasectomy is used.

The resulting transgenic mice (chimeric mice) are confirmed for theincorporation of the above DNA fragment and then crossed with wild-typemice for the birth of F1 mice. Among F1 mice that are born as a resultof this crossing, mice having the above DNA fragment (heterozygote) insomatic cells are transgenic mice capable of transmitting the above DNAfragment to germ cells.

The mouse with liver damage of the present invention may be a mouse ofany generation of the above transgenic mice, as long as the introducedDNA fragment is a heterozygote. The selection of a heterozygote can betested by screening chromosomal DNA separated and extracted from the F1mouse tail by Southern hybridization or a PCR method, for example.

Moreover, from the thus obtained transgenic mice, 2- to 3-week-oldtransgenic mice exhibiting the serum ALT (alanine aminotransferase)level of 30 (Karmen unit) or higher are selected. Preferably, 3-week-oldor 4 week-old transgenic mice exhibiting the serum ALT level of 30(Karmen unit) or higher, further preferably 6 week-old transgenic miceexhibiting the serum ALT level of 45, 50 or 55 (Karmen unit) or higher,particularly preferably 8-week-old transgenic mice exhibiting the serumALT level of 60, 65, or 70 (Karmen unit) or higher are selected. A serumALT level can be an indicator for the degree of liver damage. The higherthe serum ALT level, the higher the degree of liver damage.

The above ES cells and blastocysts to be used for preparing the mousewith liver damage of the present invention are not particularly limitedand ES cells and blastocysts from various mouse lines can be used. Forexample, cells from 129 SvEv mice, C57BL/6J mice, or the like can beused.

According to the method for preparing the mouse with liver damage of thepresent invention, the resulting mouse can have a transgene in aheterozygous form. Therefore, the transgenic mice can be efficientlyprepared in large numbers, and desired transgenic mice can be selectedand prepared efficiently from the thus obtained transgenic mice byscreening for mice having a high degree of liver damage while having theuPA gene in a heterozygous form.

Moreover, the mouse with liver damage of the present invention has mouseproductivity higher than that of conventional transgenic mice having theuPA gene in a homozygous form, since the mouse with liver damage of thepresent invention can have the uPA gene in a heterozygous form.Specifically, first, many heterozygous female mice should be produced inorder to obtain many homozygous mice. Thereafter, homozygous mice shouldbe obtained by external fertilization or natural mating of theheterozygous mice. This process requires two generations and at least 6months in total. Moreover, the thus obtained homozygous mice accountingfor only about 25% of the total number of the thus obtained mice areobtained. In contrast, many heterozygous mice can be obtained in thesecond generation (via single generation) by performing externalfertilization or natural mating with wild-type mice that can bepurchased in large numbers from breeders. The time period required forthis process is at least 3 months. Moreover, about 50% of the totalnumber of mice obtained herein are heterozygous mice, indicating that alarge number of necessary mice can be produced within a short timeperiod highly efficiently. Also, when a cross-bred line produced withanother genetically mutated mouse (e.g., gene deficiency or introducedgenes) is used for an experiment, mice that can be efficiently used forthe experiment can be obtained if heterozygous uPA transgenic mice canbe used. For example, when mice each having the introduced uPA gene in aheterozygous form and another type of gene mutation in a heterozygousform are used with each other to produce a mouse having the uPA gene andanother type of gene mutation in a double homozygous form, the thusobtained mice having both genes in a homozygous form account for only 6%of the thus obtained mice. Furthermore, female and male homozygous miceshould be obtained and then breeding and production should be performedin order to obtain a considerable number of mice to be used for anexperiment. Meanwhile, the thus obtained mice having the uPA gene in aheterozygous form and another type of gene mutation in a homozygous formaccount for about 12.5%. This indicates that mice required for anexperiment can be obtained at this time point with production efficiencyhigher than that of the production of mice having both genes in ahomozygous form. This means that a considerable number of mice that canbe used for an experiment can be obtained earlier by a single generationthan the production of mice having both genes in a homozygous form. Asdescribed above, the fact that heterozygous mice can be used enables toobtain high production efficiency, so as to contribute to save the spacefor an animal room to be used for keeping and obtaining mice necessaryfor the experiment, resulting in a shorter period required forproduction, a drastic reduction in the number of mice to be used, andthe reduction of experimenters' efforts.

In the present invention, examples of the “mouse with liver damage”include portions of the mouse. The term “a portion(s) of the mouse”refers to, mouse-derived tissues, body fluids, cells, and disruptedproducts thereof or extracts therefrom, for example (the examplesthereof are not particularly limited to them). Examples of such tissuesinclude, but are not particularly limited to, heart, lungs, kidney,liver, gallbladder, pancreas, spleen, intestine, muscle, blood vessel,brain, testis, ovary, uterus, placenta, marrow, thyroid gland, thymusgland, and mammary gland. Examples of body fluids include, but are notparticularly limited to, blood, lymph fluids, and urine. The term“cells” refers to cells contained in the above tissues or body fluids,and examples thereof include cultured cells, sperm cells, ova, andfertilized eggs obtained by isolation or culture thereof. Examples ofcultured cells include both primary cultured cells and cells of anestablished cell line. Examples of the portions of the mouse alsoinclude tissues, body fluids, and cells at the developmental stage(embryonic stage), as well as the disrupted products or extractsthereof. In addition, an established cell line from the mouse with liverdamage of the present invention can be established using a known method(Primary Culture Methods for Embryonic Cells (Shin Seikagaku Jikken Koza(New Biochemical Experimental Lecture Series), Vol. 18, pages 125-129,TOKYO KAGAKU DOZIN CO., LTD., and Manuals for. Mouse EmbryoManipulation, pages 262-264, Kindai Shuppan)).

The present invention further provides an immunodeficient mouse withliver damage. The immunodeficient mouse with liver damage of the presentinvention can be used as a host mouse for transplantation of humanhepatocytes. The immunodeficient mouse with liver damage of the presentinvention can be obtained by crossing the above mouse with liver damagewith an immunodeficient mouse.

Examples of the “immunodeficient mouse” may be any mouse that does notexhibit rejection against hepatocytes (in particular, human hepatocytes)from a different animal origin, and include, but are not limited to,SCID (severe combined immunodeficiency) mice exhibiting deficiency in T-and B-cell lines, mice (NUDE mice) that have lost T cell functionsbecause of genetic deletion of the thymus gland, and mice (RAG2 knockoutmice) produced by knocking out the RAG2 gene by a known gene targetingmethod (Science, 244: 1288-1292, 1989). A preferable example thereof isa SCID mouse.

The immunodeficient mouse with liver damage of the present invention hasa gene that specifies the phenotype of immunodeficiency in a homozygousform. The immunodeficient mouse with liver damage of the presentinvention may also have a DNA fragment containing the uPA gene from theabove mouse with liver damage in either a heterozygous form or ahomozygous form. Even when the immunodeficient mouse with liver damageof the present invention has the uPA gene in a heterozygous form, humanhepatocytes transplanted into the mouse can engraft for long periods oftime. Examples of the genotype of the immunodeficient mouse with liverdamage of the present invention include, but are not limited to, uPA(+/−)/SCID (+/+) and uPA (+/+)/SCID (+/+).

Heterozygous mice or homozygous mice can be selected by screening, asdescribed above, chromosomal DNAs separated and extracted from the tailsof the thus obtained offspring by Southern hybridization or a PCRmethod.

In the present invention, examples of the “immunodeficient mouse withliver damage” include portions of the mouse. The term “a portion of themouse” is as defined above.

Moreover, the present invention provides a chimeric mouse having humanhepatocytes. The chimeric mouse of the present invention isimmunologically deficient, which is prepared by introducing, in aheterozygous form, a DNA fragment containing cDNA that encodes anurokinase-type plasminogen activator operably linked under the controlof the liver-specific promoter and enhancer region, and has a chimericliver containing human hepatocytes.

The chimeric mouse of the present invention can be prepared bytransplanting human hepatocytes into the above immunodeficient mousewith liver damage of the present invention.

As human hepatocytes to be used for transplantation, human hepatocytesisolated from normal human liver tissue by a conventional method such asa collagenase perfusion method can be used. The thus separatedhepatocytes can also be used by thawing after cryopreservation.Alternatively, the chimeric mouse hepatocytes, which are defined as thehuman hepatocytes separated by a technique such as a collagenaseperfusion method from a chimeric mouse liver, in which mouse hepatocyteshave been replaced by human hepatocytes, can be used in a fresh state,and the cryopreserved chimeric mouse hepatocytes are also availableafter thawing.

Such human hepatocytes can be transplanted into the liver via the spleenof the above immunodeficient mouse with liver damage. Such humanhepatocytes can also be directly transplanted via the portal vein. Thenumber of human hepatocytes to be transplanted may range from about 1 to2,000,000 cells and preferably range from about 200,000 to 1,000,000cells. The gender of the immunodeficient mouse with liver damage is notparticularly limited. Also, the age on days of the immunodeficient mousewith liver damage upon transplantation is not particularly limited. Whenhuman hepatocytes are transplanted into a young mouse (early weeks ofage), human hepatocytes can more actively proliferate as the mousegrows. Hence, about 0- to 40-day-old mice after birth, and particularlyabout 8- to 40-day-old mice after birth are preferably used.

Mice after transplantation can be maintained by a conventional method.After transplantation, blood is collected periodically from the mousetail, and then the human albumin concentration in mouse blood ismeasured. Since human albumin concentration correlates with thereplacement rate of human hepatocytes in the mouse liver, the degrees ofthe engraftment and the proliferation of human hepatocytes can beinferred with the value of human albumin concentration. A mouse inferredto have a replacement rate of 70% or more on the basis of the bloodhuman albumin concentration can be used as a chimeric mouse with a highdegree of replacement for pharmacokinetic studies, infection studieswith hepatitis virus, or the like. In the case of mice, when about 1 to10×10⁵ human hepatocytes are transplanted, the mouse is maintained forabout 40 to 100 days, and thus a blood human albumin concentrationranging from 100,000 to 30,000,000 ng/mL can be obtained.

The thus transplanted human hepatocytes account for at least 10%, 20% ormore, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, 90% or more, 95% or more, or even a higher percentage ofhepatocytes in the liver of the chimeric mouse.

Transplanted human hepatocytes retain the functions and the propertiesof normal human hepatocytes in the liver of the chimeric mouse for atleast 2 weeks, 3 or more weeks, 4 or more weeks, 5 or more weeks, 10weeks, 20 weeks, 30 weeks, and 40 weeks, and most preferably for aperiod during which the mouse survives.

Examples of “the functions and the properties of human hepatocytes”include, but are not limited to, drug-metabolizing functions, proteinsynthesis, gluconeogenesis, urea synthesis, bile synthesis, lipidsynthesis, glucose metabolism, detoxication, and infectiveness againsthepatitis virus.

Transplanted human hepatocytes retain 50% or more, 60% or more, 70% ormore, 80% or more, 90% or more, 95% or more, or even a higher percentageof the functions and the properties in the normal human liver.

The present invention further provides a method for screening for asubstance that affects human liver functions, with the use of the abovechimeric mouse.

An example of the method is an evaluation method comprising thefollowing steps of:

(a) administering a test substance to the above chimeric mouse;

(b) measuring one or more values selected from the group consisting ofthe human albumin concentration, the body weight curve, theliver-weight-to-body-weight ratio, the total albumin level, the totalprotein level, the ALT level, the AST level, and the total bilirubinlevel in the chimeric mouse to which the test substance is administeredin (a); and(c) selecting a test substance that causes an increase or an decrease inany one or more of the human albumin concentration, the body weightcurve, the liver-weight-to-body-weight ratio, the total albumin level,the total protein level, the ALT level, the AST level, and the totalbilirubin level measured in (b), compared with the human albuminconcentration, the body weight curve, the liver-weight-to-body-weightratio, the total albumin level, the total protein level, the ALT level,the AST level, and the total bilirubin level of the chimeric mouse towhich no test substance is administered.

Examples of the “test substance” in the method of the present inventionare not particularly limited and include natural compounds, organiccompounds, inorganic compounds, proteins, antibodies, peptides, andsingle compounds such as an amino acid, and nucleic acids, as well ascompound libraries, expression products from gene libraries, cellextracts, cell culture supernatants, products of fermentingmicroorganisms, extracts from marine creatures, plant extracts, extractsfrom prokaryotic cells, extracts from eukaryotic single cells, andextracts from animal cells. These products may be purified products orcrude products such as plant, animal, or microbial extracts. Also, amethod for producing a test substance is not particularly limited. Atest substance to be used herein may be a substance isolated from anatural product, synthesized chemically or biochemically, or prepared bygenetic engineering techniques.

The above test substance can be adequately labeled and then used asnecessary. Examples of labels include radiolabels and fluorescentlabels. Examples of the test substance include, in addition to the abovetest samples, mixtures of a plurality of types of these test samples.

In this method, examples of a method for administering a test substanceto mice are not particularly limited. Such an administration method canbe adequately selected from among oral administration or parenteraladministration such as subcutaneous, intravenous, local, transdermal,and enteral (intrarectal) administration, depending on the type of atest substance to be administered.

The rate of replacement by human hepatocytes in the mouse liver can bepredicted by measuring the human albumin concentration in mouse blood byELISA, immunonephelometry, or the like. For prediction, a correlationcurve of human albumin concentrations and replacement rates should beprepared in advance as described below. Blood is collected before theautopsy of a chimeric mouse, and then the human albumin concentration isdetermined. Frozen sections or paraffin sections are prepared from theentire liver or partial hepatic loves collected upon autopsy.Immunostaining is performed using an antibody specific to humanhepatocytes, such as a human specific cytokeratin 8/18 (hCK8/18)antibody. Photographs of the sections are taken under a microscope, theproportion of the hCK8/18-positive area per liver section is calculatedto give a replacement rate. Human albumin concentrations and replacementrates are plotted on a graph, thereby finding a correlation equation.The human albumin concentration in mouse blood is entered to thecorrelation equation, so that a replacement rate can be roughlycalculated. Furthermore, the body weight is measured over time, thehealth status of the mouse can be predicted. A biochemical test isperformed for blood collected upon autopsy. For example, a total albuminlevel, a total protein level, and the like are measured, and thus thehealth status of the mouse can be clarified. The degree of liver damageof a chimeric mouse can be clarified by measuring the liver weight, thebody weight, ALT, AST, and the total bilirubin levels, for example.Specifically, the effects of a test substance against human hepatocytescan be determined using increases or decreases in these numericalfigures as indicators.

The present invention further provides a method for evaluatinghepatotoxicity of a test substance against human hepatocytes, with theuse of the above chimeric mouse.

An example of this method is an evaluation method comprising thefollowing steps of:

(a) administering a test substance to the above chimeric mouse;

(b) measuring one or more values selected from the group consisting ofthe human albumin concentration, the body weight curve, theliver-weight-to-body-weight ratio, the total albumin level, the totalprotein level, the ALT level, the AST level, and the total bilirubinlevel in the chimeric mouse to which the test substance is administeredin (a); and(c) evaluating the effect of the test substance on human hepatocytesusing, as an indicator, an increase or a decrease in any one or more ofthe human albumin concentration, the body weight curve, theliver-weight-to-body-weight ratio, the total albumin level, the totalprotein level, the ALT level, the AST level, and the total bilirubinlevel measured in (b), compared with the human albumin concentration,the body weight curve, the liver-weight-to-body-weight ratio, the totalalbumin level, the total protein level, the ALT level, the AST level,and the total bilirubin level of the chimeric mouse to which no testsubstance is administered.

Examples of the “test substance” and the “administration method” thereofinclude those defined above.

As described above, the degree of liver damage of a chimeric mouse canbe analyzed on the basis of human albumin concentration, body weightcurve, liver-weight-to-body-weight ratio, total albumin level, totalprotein level, ALT level, AST level, and total bilirubin level. With theuse of increases or decreases in these numerical figures as indicators,the toxicity of the test substance against human hepatocytes can bedetermined and evaluated.

The present invention further provides a method for screening for asubstance effective for treatment of viral hepatitis, with the use ofthe above chimeric mouse.

An example of this method is an evaluation method comprising thefollowing steps of:

(a) inoculating a hepatitis virus into the above chimeric mouse;

(b) administering a test substance to the chimeric mouse inoculated withthe hepatitis virus in (a);

(c) measuring one or more values selected from the group consisting ofthe human albumin concentration, the body weight curve, theliver-weight-to-body-weight ratio, the total albumin level, the totalprotein level, the ALT level, the AST level, the total bilirubin level,the viral load, and the amount of a virus-derived protein of thechimeric mouse to which the test substance is administered in (b); and(d) selecting a test substance causing a change in any one or more ofthe human albumin concentration, the body weight curve, theliver-weight-to-body-weight ratio, the total albumin level, the totalprotein level, the ALT level, the AST level, the total bilirubin level,the viral load, and the amount of a virus-derived protein measured in(c), compared with the human albumin concentration, the body weightcurve, the liver-weight-to-body-weight ratio, the total albumin level,the total protein level, the ALT level, the AST level, the totalbilirubin level, the viral load, and the amount of a virus-derivedprotein in the chimeric mouse to which no test substance isadministered.

Examples of hepatitis viruses to be used for inoculation includehepatitis type A virus, hepatitis type B virus, hepatitis type C virus,hepatitis type D virus, and hepatitis type E virus. Viruses can beinoculated via intravascular or intraperitoneal administration.

The above chimeric mouse to be used in this method is preferably a mousethat satisfies at least one of the following conditions: 3 or more weekshave passed after the transplantation of human hepatocytes; the bloodhuman albumin concentration is 1 mg/mL or higher; and human hepatocytesaccount for 10% or more of all hepatocytes.

Examples of the “test substance” and the “administration method” thereofinclude those defined above.

The degree of liver damage due to hepatitis viruses can be found on thebasis of human albumin concentration, body weight curve,liver-weight-to-body-weight ratio, total albumin level, total proteinlevel, ALT level, AST level, total bilirubin level, viral load, and theamount of a virus-derived protein. With the use of changes in thesenumerical figures as indicators, the effectiveness of a test substancein treatment of viral hepatitis can be determined and evaluated.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

Hereafter, the present invention is described in greater detail withreference to the following examples.

EXAMPLE 1 Preparation of uPA Transgenic Mice Using DNA MicroinjectionMethod

(1) Preparation of Vector Containing an uPA Gene and an Albumin Promoter

Regarding a uPA gene, total RNA was extracted from mouse liver by anAGPC method (acid-guanidinium-isothiocyanate-phenol-chloroform), andthen dissolved in RNase-free water. A reverse transcription reaction wasperformed using the above-obtained total RNA, a uPA gene-specific primer(the antisense sequence having a length from the 1341^(st) to the1360^(th) nucleotides) prepared based on the sequence of the uPA gene(Accession No.: NM008873 (SEQ ID NO: 11)) registered in the publisheddatabase, and Long Range Reverse Transcriptase (Qiagen) at 25° C. for 10minutes, and then performed at 42° C. for 90 minutes. After 5 minutes ofreverse transcriptase inactivation treatment at 85° C., RNaseH(Invitrogen) was added, the resultant was treated at 37° C. for 20minutes to digest mRNA, and thus only cDNA remained. PCR was performedusing the thus synthesized cDNA as a template. The above reactionsolution in an amount 1/10 the total amount thereof was added. As anenzyme, Phusion DNA polymerase (Fynnzymes) was used. A PCR primer (thesense sequence having a length from the 39^(th) nucleotide to the61^(st) nucleotide) was prepared based on the sequence of the uPA gene(Accession No.: NM008873). The fragment amplified by PCR has a length ofnucleotide Nos. 39-1360. The thus obtained DNA fragment was introducedinto an expression plasmid having a mouse albumin promoter/enhancerdescribed later, thereby constructing “mAlb uPAInt2.” The configurationof the “mAlb uPAInt2” gene is shown in FIG. 1. The 2nd exon, intron, andthe 3rd exon of rabbit β globin, the ORF portion of mouse uPA, and polyAsignal in the 3rd exon of rabbit β globin were ligated downstream of themouse albumin enhancer/promoter.

(2) Microinjection of DNA into the Pronuclei of Fertilized Eggs

The concentration of the DNA fragment was adjusted to 3 ng/μL, and thenthe DNA fragment was injected into pronuclear stage fertilized eggscollected from CB-17/Icr and Scid-beige cross-bred mice. DNA wasinjected into such a fertilized egg by microinjection. 635 out of 748fertilized eggs, into which DNA had been injected, survived, and 469eggs thereof differentiated into the 2-cell stage embryos. The 2-cellstage embryos were transplanted into the uterine tubes of recipient ICRmice treated in advance to be in pseudo-pregnancy. 108 offspring wereobtained. Whether or not the thus obtained offspring contained the uPAgene was analyzed by PCR (The Tokyo Metropolitan Institute of MedicalScience). As a result of PCR, it was confirmed that one mouse containedthe target DNA. A uPA transgenic mouse line was established from the onemouse.

(3) Measurement and Analysis of Serum ALT Levels in uPA Transgenic Mice

Blood was collected from the thus obtained mice having the uPA gene, soas to obtain the serum. Subsequently, the effect due to the expressionof the uPA gene in the liver, and specifically, the damage ofhepatocytes were analyzed by measuring the ALT levels. The ALT levelswere measured using “Transaminase CII-Test Wako” (Wako Pure ChemicalIndustries, Ltd., cat#431-30901). After serum collection, serum sampleswere preserved at −80° C. until measured.

The method for measuring ALT was performed on 1/20 the scale of thestandard procedure 1 included with “Transaminase CII-Test Wako”. First,a substrate enzyme solution for ALT: 10 mL of a substrate buffer for ALTwas added to 1 vial of an enzyme agent for ALT and the enzyme agent wasdissolved in the buffer. Furthermore, a chromogenic solution: 40 mL of acolor former solution was added to 1 vial of color former and the colorformer was dissolved in the solution.

Next, a CORNING 25850 96-well U-bottomed plate was prepared on ice. Aserum sample (1 μL) was added to the plate. The plate was removed fromice, 25 μL of the substrate enzyme solution was added, and then heatedat 37° C. for 5 minutes. STND (×½ dilution: 1 μL, ×1:1, 2 μL) was addedto empty wells, and then 25 μL of the chromogenic solution was added toeach well. The substrate enzyme solution (25 μL) was added to wellscontaining STND, and then heated at 37° C. for 20 minutes. A stopsolution (100 μl) was added to each well. The solution was stirred wellwith a plate mixer, and then absorbance was measured at 570 nm within 60minutes after stirring. A calibration curve was prepared using themeasurement value of STND, thereby calculating the values representingthe activity in samples.

No mouse with a high ALT level was confirmed from among mice subjectedto measurement. The uPA transgenic mouse of interest must exhibit acondition wherein the expression level of the uPA gene is optimum forthe subsequent hepatocyte transplantation. Mice exhibiting such optimumexpression level should be selected after preparation of many uPAtransgenic mice. It was revealed that this method is not a suitable fora method for probabilistically preparing such optimum mice in this case,because of the limited efficiency of the preparation of transgenic mice.

EXAMPLE 2 Preparation of uPA Transgenic Mice Via ES Cells

(1) Establishment of ES Cells Prepared by Inserting the uPA Gene

In this example, a uPA gene insertion vector “mAlb uPAInt2ES” (FIG. 2)was constructed and used. This vector was constructed by introducing aneomycin resistance gene that is expressed under the control of an SV40promoter to the “mAlb uPAInt2” plasmid in order to impart drugselectivity in ES cells. The vector was introduced by electroporationinto ES cells obtained from a 129 SvEv mouse, followed by selectiveculture using G418. The thus obtained G418-resistant colonies weresubjected to the testing by PCR for ES cells into which the gene hadbeen introduced. This is as described specifically below.

A uPA gene insertion vector (uPA) DNA (25-30 μg) was linearized bycleavage with a restriction enzyme, and then purified. The DNA wassuspended in an electroporation buffer (20 mM HEPES pH7.0, 137 mM NaCl,5 mM KCl, 6 mM D-glucose, 0.7 mM Na₂HPO₄) containing 3×10⁶ mouse EScells. Gene transfer was performed under the conditions of FieldStrength 185V/cm and Capacitance 500 μF. Selective culture was performedwith G418 (Geniticin) (SIGMA, G-9516) with a final concentration of 200μg/mL, 24 hours after gene transfer. ES cells were cultured using aDulbecco's modified Eagle's medium (DMEM) (Gibco/BRL, 11965-084) culturesolution supplemented with fetal bovine serum having a finalconcentration of 15% (Hyclone, SH30071), L-glutamine having a finalconcentration of 2 mM (Gibco/BRL, 25030-081), non-essential amino acidseach having a final concentration of 100 μM (Gibco/BRL, 11140-050),HEPES having a final concentration of 10 mM (Gibco/BRL, 15630-080),penicillin/streptomycin each having a final concentration of 100 U/mL(Gibco/BRL, 15140-122), β-mercaptoethanol having a final concentrationof 100 μM (SIGMA, M-7522), and ESGRO (LIF) having a final concentrationof 1000 U/mL (Gibco/BRL, 13275-029) (hereinafter referred to as “ESmedium”).

Moreover, as feeder cells for ES cells, MEF (Mouse Embryonic Fibroblast)cells isolated from E14.5 embryos were used. A culture solution usedherein was a DMEM (Gibco/BRL, 11965-084) culture solution supplementedwith fetal bovine serum having a final concentration of 10% (Hyclone,SH30071), L-glutamine having a final concentration of 2 mM (Gibco/BRL,25030-081), non-essential amino acids each having a final concentrationof 100 μM (Gibco/BRL, 11140-050), and penicillin/streptomycin eachhaving a final concentration of 100 U/mL (Gibco/BRL, 15140-122)(hereinafter, referred to as “MEF medium”). MEF cells cultured toconfluency in a 150-cm² flask were removed with trypsin/EDTA (0.05%/1mM, Gibco/BRL, 25300-047) and then plated again on four 10 cm dishes,two 24-well plates, two 6-well plates, six 25 cm²-flasks, and two 75 cm²flasks at optimal concentrations.

(2) Adjustment of ES Cells for Genotype Analysis

On day 5 after gene transfer, G418-resistant colonies that had appearedwere passaged to a 24-well plate, as described below. Specifically,G418-resistant colonies were transferred to a 96-well microplatecontaining 150 μL, of a trypsin/EDTA solution using Pipetman (Gilson).After 20 minutes of treatment within an incubator at 37° C., pipettingwas performed with Pipetman to obtain single cells. The cell suspensionwas transferred to a 24-well plate and then culture was continued. Twodays later, cells on the 24-well plate were divided into two groups,cells for cryopreservation and cells for DNA extraction. Specifically,500 μL of trypsin/EDTA was added to cells, cells were treated for 20minutes within an incubator at 37° C., and then 500 μL of ES medium wasadded. Gentle pipetting was performed with Pipetman, so as to obtainsingle cells. Subsequently, a half of the cell suspension wastransferred to a 24-well plate containing 1 mL of ES medium. One mL ofES medium was also added to the original 24-well plate. Two days later,the medium in one of the 24-well plates was removed. 1 mL of medium forfreezing prepared by adding fetal bovine serum having a finalconcentration of 10% and dimethyl sulfoxide (DMSO) having a finalconcentration of 10% (Sigma, D-5879) to an ES medium was added. Theresultant was sealed and then cryopreserved at −70° C.

ES cells into which the gene had been introduced were tested by PCR asdescribed below. Specifically, medium was removed from each well of the24-well plates in which cells had grown to confluency. After washingwith PBS, 250 μL of a dissolution buffer (1% SDS, 20 mM EDTA, 20 mM TrispH7.5) and 5 μL of proteinase K (20 mg/mL) were added and then theresultant was shaken well, followed by heating at 52° C. fordissolution. DNA was extracted from a dissolved sample byphenol/chloroform extraction, and then the resultant was used astemplate DNA for PCR.

(3) Method for Analyzing the Genotype of ES Cells: ES Cells into whichthe uPA Gene Had been Introduced were Selected by the FollowingProcedure.

PCR primers used herein were set in rabbit β globin. The sequences are:a sense primer: GGGCGGCGGTACCGATCCTGAGAACTTCAGGGTGAG (SEQ ID NO: 1) andan antisense primer: GGGCGGCGGTACCAATTCTTTGCCAAAATGATGAGA (SEQ ID NO:2). Reaction was performed according to the method included withAmpiTaqGold (ABI). After 9 minutes of activation of the enzyme at 95°C., the cycle of PCR [94° C. for 30 seconds (denaturation), 63° C. for30 seconds (annealing), and 72° C. for 1 minute (extension)] wasrepeated 40 times. After the completion of the reaction, the reactionsolution was subjected to 2% agarose gel electrophoresis, so as toconfirm PCR products.

Clones for which gene transfer had been confirmed by PCR analysis werethawed by heating the previously cryopreserved 96-well plate to 37° C.,and then passaged to a 24-well plate. Clones in the 24-well plate werecultured for 24 hours at 37° C., medium exchange was performed to removeDMSO and liquid paraffin. When each clone reached 75% to 90% confluency,respectively, clones were passaged from the 24-well plate to a 6-wellplate. Moreover, when clones that had grown to 75% to 90% confluencywere obtained in 2 wells of the 6-well plate, clones in one well werecryopreserved and clones in the other well were used for injection intoblastocysts and DNA extraction.

Cryopreservation was performed as follows. Specifically, cells wererinsed twice with PBS, 0.5 mL of Trypsin was added, and then thetemperature was kept at 37° C. for 15 to 20 minutes to perform trypsintreatment. Furthermore, 0.5 mL of ES cell medium was added, pipettingwas performed 35 to 40 times, and thus the mass of ES cells wascompletely dissociated. The cell suspension was transferred to a 15 mLcentrifugal tube. Wells were further washed with 1 mL of ES cell medium,and then the resultants were collected in a tube. The tube wascentrifuged at 1,000 rpm for 7 minutes. The medium was removed and thensuspended again in 0.25 mL of ES cell medium. 0.25 mL of 2× frozenmedium was added. The contents of the wells were transferred to acryogenic vial, frozen at −80° C., and then preserved in liquidnitrogen.

Regarding cells for injection into blastocysts and DNA extraction, themass of ES cells was completely dissociated, one-quarter thereof wasused for injection into blastocysts, one-third of the remaining cellsand two-third of the same were each passaged into a 60 mm dish coatedwith gelatin. When the former cells grew to confluency, genomic DNA forPCR analysis was extracted. When the latter cells grew to confluency,the cells were divided into three groups and then frozen.

(4) Preparation of Chimeric Mice Using ES Cells Having the uPA Gene

Regarding ES cell clones for which gene transfer had been confirmed,chimeric embryos were prepared using the blastocysts of C57BL/6J mice ashost embryos. The chimeric embryos were each transplanted into theuterine horn of a pseudopregnant mouse to obtain offspring. Host embryoswere collected by perfusion of the uterine tube and the uterus withWhitten's medium supplemented with 100 μM trypsin/EDTA on day 3 ofpregnancy. 8-cell-stage embryos or morulae were cultured for 24 hours inWhitten's medium. The thus obtained blastocysts were used for injection.ES cells used for injection were dispersed by TE treatment on day 2 or 3of passage, and then left to stand at 4° C. until the micromanipulationof these cells. As a pipette for injection of ES cells, glass capillarytubing (Sutter, inner diameter of about 20 μm) was used. A pipette forholding embryos used herein was processed as follows. A glassmicrotubule with an outer diameter of 1 mm (NARISHIGE) was pulled thinusing a micropipette puller (Sutter, P-97/IVF), and then its tip with anouter diameter ranging from 50 μm to 100 μm was cut using a microforge(De Fonburun), and then processed to have an aperture of 10 μm to 20 μm.The pipette for injection and the pipette for holding were connected toa micromanipulator (Lica) with a piezo system (Primetech PAMS-CT150)connected thereto. As a chamber used for micromanipulation, perforatedslide glass to which cover glass had been adhered with bees wax wasused. Two drops of Hepes-buffered Whitten's medium supplemented withabout 10 μL of 0.3% BSA were placed thereon, and then the top face wascovered with mineral oil (Sigma). One drop contained about 100 ES cells,and the other drop contained about 20 expanded blastocysts. About 15 EScells were injected per embryo. Micromanipulation was always performedunder an inverted microscope. Manipulated embryos were transplanted intothe uterine horns of recipient ICR female mice on day 2 of pseudopregnancy. Recipient female mice that had not delivered offspring evenon the predicted delivery date were subjected to cesarean section. Theresulting offspring were raised by surrogate parents. As a result ofinjection of 45 clones of ES cells into the blastocysts of C57BL/6Jmice, male chimeric mice were obtained from 39 clones.

(5) Test of the Transmission of the uPA Gene to the Germ Line

Chimeric mice were crossed with C57BL/6J mice, and then whether or notES-cell-derived offspring were obtained was tested. If the germ cells ofthe chimeric mice were derived from ES cells, the thus deliveredoffspring would have wild-type hair color. If the germ cells of thechimeric mice were derived from the blastocysts of C57BL/6J mice, thethus delivered offspring would have black hair color. As a result ofcrossing, offspring having wild-type hair color were delivered in 25lines, and thus the transmission of ES cells to the germ line wasconfirmed.

Next, DNA was extracted from the tail portions of these mice havingwild-type hair color and then subjected to PCR to examine if the uPAgene had been transmitted. As a result, the transmission of the uPA genewas confirmed for ES-cell-derived offspring of 14 lines.

(6) Measurement and Analysis of Serum ALT Levels in uPA Transgenic Mice

Blood was collected from the thus obtained mice having the uPA gene, soas to obtain the serum. Subsequently, the effect due to the expressionof the uPA gene in the liver, and specifically, the damage ofhepatocytes were analyzed by measuring the ALT levels. The ALT levelswere measured using “Transaminase CII-Test Wako” (Wako Pure ChemicalIndustries, Ltd., cat#431-30901). After serum collection, serum sampleswere preserved at −80° C. until measured.

The method for measuring ALT was performed on 1/20 the scale of thestandard procedure 1 included with “Transaminase CII-Test Wako”. First,a substrate enzyme solution for ALT: 10 mL of a substrate buffer for ALTwas added to 1 vial of an enzyme agent for ALT and the enzyme agent wasdissolved in the buffer. Furthermore, a chromogenic solution: 40 mL of acolor former solution was added to 1 vial of color former and the colorformer was dissolved in the solution.

Next, a CORNING 25850 96-well U-bottomed plate was prepared on ice. Aserum sample (1 μL) was added to the plate. The plate was removed fromice, 25 μL of the substrate enzyme solution was added, and then heatedat 37° C. for 5 minutes. STND (×½ dilution: 1 μL, ×1:1, 2 μL) was addedto empty wells, and then 25 μL of the chromogenic solution was added toeach well. The substrate enzyme solution (25 was added to wellscontaining STND, and then heated at 37° C. for 20 minutes. A stopsolution (100 μl) was added to each well. The solution was stirred wellwith a plate mixer, and then absorbance was measured at 570 nm within 60minutes after stirring. A calibration curve was prepared using themeasurement value of STND, thereby calculating the values representingthe activity in samples.

Of 14 mouse lines measured, 3 lines of heterozygous mice were confirmedto have high ALT levels (FIG. 3).

The following experiment of human hepatocyte transplantation wasperformed using 2 lines (#1C2 and #2C7) of mice with high ALT levelsfrom among the thus obtained 3 lines.

EXAMPLE 3 Preparation of Chimeric Mice

(1) Immunodeficient Mice with Liver Damage

uPA-Tg mice (hemizygote, +/−) prepared in Example 2 above wereback-crossed twice with SCID-bg mice, thereby obtaining mice having thegenotype of uPA-Tg(+/−)SCID(+/+). Sperm cells were collected from themale mice, external fertilization was performed with unfertilized eggsof SCID mice (homozygote, +/+), and then the fertilized eggs werereturned into surrogate mother mice. Among offspring delivered, micehaving a Tg gene therein were selected and then subjected to naturalmating, so that mice having both genotypes (uPA-Tg(+/−)/SCID (+/+)) wereobtained. uPA-Tg (+/−) and uPA-Tg(−/−) were distinguished from eachother by a genome PCR method using sequences specific to the transgeneas primers.

Forward primer (SEQ ID NO: 3) 5′-GGGCGGCGGTACCGATCCTGAGAACTTCAGGGTGAG-3′Reverse primer (SEQ ID NO: 4) 5′-GGGCGGCGGTACCAATTCTTTGCCAAAATGATGAGA-3′

In addition, SCID (+/+), SCID (+/−), and SCID(−/−) were distinguishedfrom each other by a PCR-RFLP method.

Next, the thus obtained uPA-Tg(+/−)/SCID(+/+) mice were crossed eachother, thereby obtaining uPA-Tg(+/+)/SCID(+/+) anduPA-Tg(+/−)/SCID(+/+). uPA-Tg(+/+) and uPA-Tg(+/−) were distinguishedfrom each other by a Southern blotting method. About 5-mm tail portionswere cut from 8- to 10-day-old mice, and then solubilized with a 3 SDS,proteinase K solution. Protein components mixed therein were removed byphenol and chloroform extraction. RNA mixed therein was denatured usingDNAse-free RNase A, and then macromolecular genomic DNA was precipitatedby isopropanol precipitation. The above genomic DNA was washed with 70%ethanol, air dried, and then dissolved again in TE. Genomic DNAextracted from a specimen, positive control genomic DNA, and negativecontrol genomic DNA (5 μg each) were completely digested with EcoRI. Thethus generated DNA fragments were separated by agarose electrophoresis,and then transferred to a nylon membrane. A DNA fragment appropriate asa probe for Southern hybridization was purified (379 bp) from uPA cDNAprobe/TA using restriction enzyme EcoRI. The above DNA fragment waslabeled with [32P] by a random prime method. The DNA fragmenttransferred to the nylon membrane was hybridized with the RI-labeled uPAcDNA probe. Non-specifically bound probes were removed by washing.Radioactive signals from the foreign gene introduced in mAlb-uPA-Int2 Tgmouse candidates were exposed to an X-ray film and thus detected.Wild-type-locus-derived 1.5-kb specific signals and mutant-locus-derived0.4 kb (wt: 1.5 kb) specific signals were detected, thereby determiningthe genotype of individual mAlb-uPA-Int2 Tg mice.

(2) Transplantation of Human Hepatocytes

As human hepatocytes, hepatocytes (Lot No.BD85, boy, 5 years old)purchased from BD Gentest were used. The cryopreserved hepatocytes werethawed and used according to a conventionally known method (Chise Tatenoet al, Near-completely humanized liver in mice shows human-typemetabolic responses to drugs. Am. J Pathol 165: 901-912, 2004).

2- to 4-week-old 7 #1C2 homozygous, 4 #1C2 heterozygous, 4 #2C7homozygous, and 7 #2C7 heterozygous uPA-Tg/SCID mice wereether-anesthetized. An about 5-mm incision was made in a flank, and then2.5×10⁵ human hepatocytes were injected via the inferior splenic pole.The spleen was returned to the peritoneal cavity and then the site wassutured. One #1C2 homozygous mouse died on day 30 of transplantation.

2 μL of blood was collected from mouse tail vein on weeks 3 and 6 aftertransplantation and then every week, and then added to 200 μL ofLX-Buffer. Human albumin concentrations in mouse blood were measured byimmunonephelometry using an autoanalyzer JEOL BM6050 (JEOL Ltd.). As aresult, increases in human albumin concentration were observed for #1C2homozygous, #1C2 heterozygous, and #2C7 homozygous mice. Specifically,mice with human albumin concentrations higher than 7 mg/mL were observed(FIG. 4 and FIG. 5). No increase in human albumin concentration wasobserved for #2C7 heterozygous mice (FIG. 5). A smooth gain in bodyweight was observed for all mice. The body weights of most #2C7heterozygous mice were at high levels (FIG. 5):

Chimeric mice (13- to 15-week-old) were anatomized 10 to 12 weeks aftertransplantation, and then liver and blood were collected. The frozensections of 7 liver lobes were prepared, and then immunostaining wasperformed using a human-specific cytokeratin 8/18 (hCK8/18) antibody(FIG. 6). The hCK8/18 positive area per area of a frozen section wasdetermined, thereby obtaining a replacement rate. As a result, #1C2homozygous mice and #2C7 homozygous mice having a replacement rate of70% or more were confirmed (FIG. 7). #1C2 heterozygous mice having areplacement rate of 60% or more were observed (FIG. 7). A correlationbetween human albumin concentrations in mouse blood and replacementrates was confirmed in a manner similar to those in conventionally knownchimeric mice (Chise Tateno et al, described above) (FIG. 7). In termsof correlation, no clear difference was confirmed between #1C2 and #2C7,and, between heterozygous mice and homozygous mice (FIG. 7).

Human hepatocytes were transplanted into 2- to 4-week-old 28 #1C2homozygous, 28 #1C2 heterozygous, 18 #2C7 homozygous, and 15 #2C7heterozygous uPA-Tg/SCID mice. As a result, chimeric mice having highreplacement rates, wherein the human albumin concentration in mouseblood was 7 mg/mL or higher, were #1C2 homozygous mice (61%), #1C2heterozygous mice (36%), #2C7 homozygous mice (28%), and #2C7heterozygous mice (0%) (FIG. 8-1, 2, FIG. 9-1, 2, FIG. 10, and FIG. 11).

Several 14- or 15-week-old mice (6 #1C2 homozygous mice, 6 #1C2heterozygous mice, and 4 #2C7 homozygous mice) were inoculated with HBVor HCV (1×10⁴ copies/mouse) via orbital venous plexus (FIG. 8-1, 2, 9-1,2). On week 1 after inoculation, blood was collected via orbital venousplexus every week, HBV and HCV viral titers were determined by areal-time quantitative PCR method and a real-time quantitative RT-PCRmethod.

RNA was extracted from 5 μL of serum collected from each mouseinoculated with HCV using SepaGene RV-R (Sanko Junyaku Co., Ltd.(currently, EIDIA Co., Ltd.), Tokyo, Japan). RNA was dissolved in 10 μLof Nuclease-free water (Life Technologies Corporation, Carlsbad, Calif.,USA) containing 1 mM DTT (Promega, Tokyo, Japan) and 0.4 U/μLribonuclease inhibitor (Takara Bio Inc. Shiga, Japan). The thusdissolved RNA was preserved at −80° C. until the quantification of serumHCV RNA level.

TaqMan EZ RT-PCR Core Reagents (Life Technologies Corporation) and 2.5μL of a dissolved undiluted RNA solution or diluted RNA solution wereused for a PCR solution. PCR was performed under conditions of 50° C.for 2 minutes (Uracil-N-Glycosylase treatment)→60° C. for 30 minutes(reverse transcription reaction)→95° C. for 5 minutes (PCR initialactivation)→[95° C. for 20 seconds (denaturation)→62° C. for 1 minute(annealing extension reaction)]×50 cycles. Reaction and analyses wereperformed using ABI Prism 7500 (Life Technologies Corporation). Primersand a probe used herein are as follows.

Forward primer: (SEQ ID NO: 5) 5′-CGGGAGAGCCATAGTGG-3′ Reverse primer:(SEQ ID NO: 6) 5′-AGTACCACAAGGCCTTTCG-3′ Probe: (SEQ ID NO: 7)5′-CTGCGGAACCGGTGAGTACAC-3′ (5′-end: FAM, 3′-end: TAMRA)

As an HCV RNA standard, serum obtained from HCV-infected chimeric micewas used. The serum had been subjected to the determination of the HCVRNA level based on artificial HCV RNA, and preserved at −80° C. untilthe quantification of serum HCV RNA. When serum HCV RNA concentrationswere measured, RNA was extracted from the serum, subjected to 10-foldserial dilution for use as the HCV RNA standard. The determination limitof the determination of serum HCV RNA using the HCV RNA standard rangesfrom 2.1×10⁴ copies/mL to 2.1×10⁷ copies/mL in serum.

DNA was extracted from 10 μL of serum collected from mice inoculatedwith HBV using SMI TEST EX-R&D (Code: R-35, Medical and BiologicalLaboratories Co., Ltd., Nagano, Japan). DNA was dissolved in 20 μL ofNuclease-free water. The thus dissolved DNA was preserved at −20° C. orlower until the quantification of HBV DNA.

5 μL of dissolved DNA and TaqMan PCR Core Reagents Kit with AmpliTaqGold (Applied Biosystems, Tokyo, Japan) were used for a PCR solution.PCR was performed under conditions of 50° C. for 2 minutes (primerannealing)→95° C. for 10 minutes (PCR initial activation)→[95° C. for 20seconds (denaturation)→60° C. for 1 minute (annealing⋅extensionreaction)]×53 cycles. Reaction was performed using ABI Prism 7500(Applied Biosystems, Tokyo, Japan). For analysis, each HBV DNA levelcalculated herein was the average level of the levels found from twowells. In addition, the thus calculated HBV DNA levels of higher than 0and less than 4.0×10⁴ copies/mL were considered to be PCR positive. Thethus calculated HBV DNA levels of 0 were considered to be PCR negative.Specifically, when both the levels found from 2 wells subjected tomeasurement were PCR positive, the result was denoted as “+” (HBVpositive). When both the levels found from two wells were PCR negative,the result was denoted as “−” (HBV negative). When one of the levelsfound from two wells was positive, the result was denoted as “±” (HBVfalse-positive).

Primers and a probe used herein had the following sequences.

Forward primer: (SEQ ID NO: 8) 5-CACATCAGGATTCCTAGGACC-3 Reverse primer:(SEQ ID NO: 9) 5-AGGTTGGTGAGTGATTGGAG-3 Probe: (SEQ ID NO: 10)5-CAGAGTCTAGACTCGTGGTGGACTTC-3 (5′-end: FAM, 3′-end: TAMRA)

As an HBV standard, a plasmid, into which an HBV gene (nucleotides 1 to2182) had been inserted and the copy number of which had been calculatedin terms of the concentration at OD260 nm, was used. The determinationlimit for the determination of serum HBV DNA ranged from 4.0×10⁴copies/mL to 4.0×10⁹ copies/mL. Specimens exhibited 4.0×10⁹ copies/mL ormore were diluted and then deremination was performed with themeasurement range.

As a result, HBV infection and HCV infection were confirmed for all theinfected mice (FIG. 8-1, 2, FIG. 9-1, 2). HCV was detected on week 1after inoculation for 7 out of 8 cases of HCV-inoculated mice, and theHCV level reached a plateau around week 4 after inoculation (FIG. 8-1,FIG. 9-1). HCV was detected on week 4 after inoculation for 1 out of 8cases (FIG. 8-1, FIG. 9-1). Regarding HBV, HBV was detected in mouseserum on week 3 after inoculation for 7 out of 8 cases, and all the micewere infected on week 4. The level reached plateau on week 8 afterinoculation (FIG. 8-2, FIG. 9-2). In terms of infection efficiency andviral growth rate, no clear difference was confirmed between #1C2 and#2C7, and, between #1C2 heterozygous and #1C2 homozygous mice (FIG. 8-1,2, FIG. 9-1, 2). In addition, in the case of #2C7 heterozygous mice,chimeric mice having high replacement rates; that is, human albuminconcentrations in mouse blood of 7 mg/mL or higher, were not obtained.Hence, none of these mice were used for the infection experiment.

The remaining mice (22 #1C2 homozygous mice, 22 #1C2 heterozygous mice,14 #2C7 homozygous mice, and 15 #2C7 heterozygous mice) were maintaineduntil they were 30 weeks old. Human albumin concentration was measuredonce a week. These mice were anatomized on week 30, and the replacementrates in liver were similarly determined. The human albuminconcentrations were found to continuously increase even after the micewere 14 weeks old or older (FIG. 10 and FIG. 11). Until week 30, nodecrease in human albumin concentration in mouse blood was observed formost of these mice. A correlation between human albumin concentrationsand replacement rates was observed at autopsy (FIG. 7).

INDUSTRIAL APPLICABILITY

According to the present invention, mice with liver damage having a highdegree of damage against mouse's original hepatocytes, while having theuPA gene in a heterozygous form, and a method for efficiently preparingthe mice with liver damage can be provided.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

The invention claimed is:
 1. A chimeric mouse, which has a chimericliver containing human hepatocytes, wherein human hepatocytes accountfor at least 10% of all hepatocytes in the chimeric liver, obtained by:transforming mouse ES cells with a DNA fragment containing aliver-specific promoter/enhancer and cDNA that encodes uPA operablylinked under the control thereof; injecting the transformed mouse EScells obtained above into a host embryo; transplanting the host embryoobtained above via the injection of the ES cells into the uterus of asurrogate mother mouse, so as to obtain a chimeric mouse; crossing thechimeric mice obtained above, so as to obtain a transgenic mouse withliver damage in which the DNA fragment is introduced in a heterozygousform, wherein the serum ALT level increases at least from when it is 6weeks old to when it is 8 weeks old; crossing the mouse with liverdamage with an immunodeficient mouse forming an immunodeficient mousewith liver damage having the uPA gene in a heterozygous form; andtransplanting human hepatocytes into the immunodeficient mouse withliver damage having the uPA gene in a heterozygous form.
 2. A chimericmouse, which has a chimeric liver containing human hepatocytes, whereinthe blood human albumin concentration is 1 mg/mL or higher, obtained by:transforming mouse ES cells with a DNA fragment containing aliver-specific promoter/enhancer and cDNA that encodes uPA operablylinked under the control thereof; injecting the transformed mouse EScells obtained above into a host embryo; transplanting the host embryoobtained above via the injection of the ES cells into the uterus of asurrogate mother mouse, so as to obtain a chimeric mouse; crossing thechimeric mice obtained above, so as to obtain a transgenic mouse withliver damage in which the DNA fragment is introduced in a heterozygousform, wherein the serum ALT level increases at least from when it is 6weeks old to when it is 8 weeks old; crossing the mouse with liverdamage with an immunodeficient mouse forming an immunodeficient mousewith liver damage having the uPA gene in a heterozygous form; andtransplanting human hepatocytes into the immunodeficient mouse withliver damage having the uPA gene in a heterozygous form.